Rutin synthase in fava d’anta: purification and influence of stressors

The flavonoid rutin is synthesized in plants from quercetin, via a process in which isoquercitrin is an intermediary metabolite. In this work, the activities of isoquercitrin synthase and rutin synthase, and the quercetin, isoquercitrin and rutin contents of fava d’anta plants stressed for water (drought and flooding) and salt (NaCl) were studied. In general, stress increased the contents of the three compounds and both enzyme activities. Semi-purified rutin synthase and isoquercitrin synthase showed Km values of 1.816 and 2.10 µM, respectively, with optimum reaction pHs of 5 and 7, respectively, and an optimum reaction temperature of 35°C. Rutin synthase was purified from leaf buds and showed an apparent molecular mass of 39 KDa by SDS-PAGE. Mass spectrometry analysis of the purified protein did not reveal any similarity to the few known sequenced glycosyltransferases.Key words: Dimorphandra mollis, faveiro, flavonoid, isoquercitrin, quercetin.

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Effect of Ocular Hypertension on D-β-Aspartic Acid-Containing Proteins in the Retinas of Rats

Journal of Ophthalmology ◽

10.1155/2019/2431481 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8 ◽

Cited By ~ 5

Author(s):

Takashi Kanamoto ◽

Takashi Tachibana ◽

Yasushi Kitaoka ◽

Toshio Hisatomi ◽

Yasuhiro Ikeda ◽

...

Keyword(s):

Mass Spectrometry ◽

Ocular Hypertension ◽

Aspartic Acid ◽

Atp Synthase ◽

Wistar Rats ◽

Protein Band ◽

Mass Spectrometry Analysis ◽

Liquid Chromatography Mass Spectrometry ◽

Spectrometry Analysis ◽

Sds Page

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

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Gel Electrophoresis/Electroelution Sorting Fractionator combined with Filter Aided Sample preparation (FASP) for deep proteomic analysis

10.1101/2021.04.16.440150 ◽

2021 ◽

Author(s):

Yassel Ramos ◽

Alexis Almeida ◽

Jenis Carpio ◽

Arielis Rodríguez-Ulloa ◽

Yasser Perera ◽

...

Keyword(s):

Mass Spectrometry ◽

Sample Preparation ◽

Protein Identification ◽

Protein Extraction ◽

Complex Mixture ◽

Fold Increase ◽

Mass Spectrometry Analysis ◽

Protein Fractionation ◽

Spectrometry Analysis ◽

Sds Page

AbstractSample preparation and protein fractionation are important issues in proteomic studies in spite of the technological achievements on protein mass spectrometry. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is SDS-PAGE-based protein fractionation due to its extraordinary resolution and the effectiveness of SDS as a solubilizing agent. Its main limitation lies in the poor recovery of the gel-trapped proteins, where protein electro-elution is the most successful approach to overcome this drawback. We created a device to separate complex mixture of proteins and peptides (named “GEES fractionator”) that is based on the continuous Gel Electrophoresis/Electro-elution Sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electro-eluted to the solution containing wells. The performance of the device was studied for SDS-PAGE-based protein fractionation in terms of reproducibility, protein recovery and loading capacity. In the SDS-free PAGE setup, complex peptide mixtures can also be fractionated. More than 11 700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the GEES fractionator combined with the Filter Aided Sample Preparation (FASP) method and mass spectrometry analysis. GEES-based proteome characterization shows a 1.7 fold increase in the number of identified proteins compared to the unfractionated sample analysis. Proteins involved in the co-regulated transcription activity, as well as cancer related pathways such as apoptosis signaling, P53 and RAS pathways are more represented in the protein identification output of GEES-based fractionation approaches.

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Mass Spectrometry Analysis and Biological Characterization of the Predatory Ant Odontomachus monticola Venom and Venom Sac Components

Toxins ◽

10.3390/toxins11010050 ◽

2019 ◽

Vol 11 (1) ◽

pp. 50 ◽

Cited By ~ 5

Author(s):

Naoki Tani ◽

Kohei Kazuma ◽

Yukio Ohtsuka ◽

Yasushi Shigeri ◽

Keiichi Masuko ◽

...

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Molecular Mass ◽

Biogenic Amines ◽

Transcriptome Analysis ◽

Mass Spectrometry Analysis ◽

Biological Characterization ◽

Spectrometry Analysis ◽

Dipeptidyl Peptidases

We previously identified 92 toxin-like peptides and proteins, including pilosulin-like peptides 1–6 from the predatory ant Odontomachus monticola, by transcriptome analysis. Here, to further characterize venom components, we analyzed the venom and venom sac extract by ESI-MS/MS with or without trypsin digestion and reducing agent. As the low-molecular-mass components, we found amino acids (leucine/isoleucine, phenylalanine, and tryptophan) and biogenic amines (histamine and tyramine) in the venom and venom sac extract. As the higher molecular mass components, we found peptides and proteins such as pilosulin-like peptides, phospholipase A2s, hyaluronidase, venom dipeptidyl peptidases, conotoxin-like peptide, and icarapin-like peptide. In addition to pilosulin-like peptides 1–6, we found three novel pilosulin-like peptides that were overlooked by transcriptome analysis. Moreover, pilosulin-like peptides 1–6 were chemically synthesized, and some of them displayed antimicrobial, hemolytic, and histamine-releasing activities.

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Extraction, Isolation and Identification of Antimicrobial Substances from Bacillus amyloliquefaciens CMN1308

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha45110654 ◽

2017 ◽

Vol 45 (1) ◽

pp. 308-315

Author(s):

Yingyou FANG ◽

Linling LI ◽

Yongliang ZHENG ◽

Honghui YUAN ◽

Xuehua ZHANG ◽

...

Keyword(s):

Mass Spectrometry ◽

Molecular Weight ◽

Bacillus Amyloliquefaciens ◽

Protein Characterization ◽

Mass Spectrometry Analysis ◽

Antimicrobial Protein ◽

Isolation And Identification ◽

Spectrometry Analysis ◽

Antimicrobial Substances ◽

Sds Page

Four separation methods of antimicrobial substances produced by CMN1308 (Bacillus amyloliquefaciens) were evaluated and selected according to number of antimicrobial substances and its activity in vitro. The results showed that extraction by acid precipitation of the fermentation supernatant of CMN1308 was the best with a diameter of inhibition zone of pathogen fungi P. expansum of 12.3 mm in a laboratory bioassay. Applying a silica thin layer chromatography (TLC), SDS-PAGE and other separation technologies we isolate antimicrobial substances, and the separated band were cut off for mass spectrometry analysis. The TLC of crude extract of CMN1308 show a topical band corresponding with the surfactin standard (Rf value =0.75), proved that the strain CMN1308 can produce this surface active compound. The mycoprotein extracted from CMN1308 was separated by Tricine-SDS-PAGE modified with the addition of urea in the separation gel. After mass spectrometric analysis and protein characterization, the isolated mycoprotein showed a maximum ion peak at M/Z of 2679 and molecular weight of 29.5 kDa, matching with protein flagellin. The extracellular antimicrobial protein of strain CMN1308 display four bands after urea-Tricine-SDS-PAGE, but after mass spectrometry analysis only two bands were identified. Band “A” with a maximum ion peak at M/Z of 1926 and molecular weight of 49.8 kDa, aligned with NCBI database, matching with DLDH (dihydrolipoamide dehydrogenase enzyme). Band “D” show the maximum ion peak at M/Z of 2936 and molecular weight of 22.4 kD, matching with a chitin binding protein. Thus, the strain CMN1308 has the potential to be developed as a commercial biological control agent for chestnut common pathogenic fungi.

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Characterization and Mass Spectrometry Analysis of Aminopeptidase N from Pseudomononas putida Lup.

Polish Journal of Microbiology ◽

10.33073/pjm-2013-046 ◽

2013 ◽

Vol 62 (4) ◽

pp. 337-343

Author(s):

Urszula Jankiewicz ◽

Maria Swiontek-Brzezinska ◽

Ewa Beata Górska ◽

Paweł Kowalczyk

Keyword(s):

Mass Spectrometry ◽

Pseudomonas Putida ◽

Gel Permeation Chromatography ◽

Aminopeptidase N ◽

Mass Spectrometry Analysis ◽

High Similarity ◽

Spectrometry Analysis ◽

Protein Substrates ◽

Sds Page ◽

Gel Permeation

An intracellular aminopeptidase N synthesized by Pseudomonas putida Lup was purified and characterized. The approx. 150-fold purified enzyme showed highest activity against A-beta-naphthylamide at pH 7.5 and at temperature 40 degrees C and was 100% thermostable for 240 min at 40 degrees C. P putida lup aminopeptidase N is a monomer with molecular mass approx. 99 kDa determined by SDS-PAGE and gel permeation chromatography. The enzyme has broad substrate specificity, but is the most active against protein substrates with N-terminal alanine and arginine. The activity of P. putida Lup aminopeptidase N is strongly inhibited in the presence of specific metallopeptidase inhibitors and is partly recovered in the presence of Zn2+ and Co2+ ions. Co2+, Mg2+ and Ca2+ ions increased the activity of the enzyme. Moreover, the enzyme was inhibited by inhibitors of cysteine enzymes. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to PepN of Pseudomonas putida GB-1.

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